CD8 expressing T cells (TCD8+) play a critical role in eradicating intracellular parasites such as viruses. TCD8+ recognize MHC class I molecules in a complex with peptides of 8 to 10 residues derived from viral proteins located in the cytosol. There is tremendous interest in the mechanism by which peptides are generated. There is indirect evidence that implicates proteasomes in the generation of antigenic peptides. Proteasomes are abundant macromolecular structures present in the cytosol and nucleus in cells with multiple protease activities. They are thought to be responsible for the energy dependent proteolysis in which ubiquitin plays a prominent role in targeting proteins for destruction. Although it is believed that at least some proteolysis occurs in the cytosol, it is uncertain whether the ultimate determinants are generated in the cytosol, or whether additional trimming occurs following transport from the cytosol. We have initiated several approaches to assess the site and nature of proteolytic mechanisms utilized in the generation of antigenic peptides. First, we produced vaccinia recombinants expressing a form of ubiquitin altered to interfere with ubiquitin-targeted proteolysis. Second, we engineered a cytosolic form of class I molecule to that should serve as a probe for the presence of antigenic peptides in the cytosol.